Construction method of ischemic nerve injury animal model
By constructing an ischemic nerve injury model using zebrafish juveniles and cobalt chloride solution, and combining it with a multi-dimensional assessment system, the problems of high cost, long cycle and single assessment system of existing models are solved, thus achieving efficient nerve injury research and drug screening.
Patent Information
- Application Number
- CN202511459917.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-10-13
- Publication Date
- 2026-02-13
AI Technical Summary
Existing animal models of ischemic nerve injury suffer from problems such as high cost, long experimental cycle, limited evaluation system and difficulty in high-throughput application, especially in mouse surgical models.
Using zebrafish juveniles as the research subject, ischemic neurological injury was induced by treating them with 1 mmol/L cobalt chloride aqueous solution for 24 h, and a multi-dimensional assessment system was constructed by combining behavioral tests, brain H&E staining and red fluorescent labeling of erythrocytes.
It significantly reduces experimental and time costs, improves the survival rate of experimental animals, and greatly enhances the repeatability and reliability of models, providing an efficient tool for high-throughput screening and efficacy evaluation of neuroprotective drugs.
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Abstract
Description
TECHNICAL FIELD
[0001] The application relates to a method for constructing an ischemic nerve injury animal model and belongs to the technical field of animal disease model construction. BACKGROUND
[0002] Ischemic nerve injury is a common nervous system pathological problem, and the core cause thereof is that insufficient blood supply (such as blood vessel obstruction and decreased perfusion pressure) of the brain leads to ischemia and hypoxia of nerve tissues. The state is continuously developed, which further causes neuron cell death and finally causes nerve dysfunction, and affects normal physiological activities of animals or human bodies. In clinical practice, ischemic nerve injury is the core pathological process of high-incidence diseases such as cerebral apoplexy and brain injury after cardiac arrest, has a high disability rate and mortality, and poses a serious threat to human health. Therefore, research on ischemic nerve injury has been a key direction in the field of biological medicine.
[0003] The pathogenesis research, drug screening and treatment scheme development of ischemic nerve injury all depend on reliable animal models. At present, the mouse is mainly used to construct a model through a surgical method in the research field of ischemic nerve injury, but the traditional method has many limitations: the specific blood vessels of the mouse need to be accurately blocked during the operation, the operation technique is very strict, and ordinary researchers are difficult to quickly master; the mortality of the mouse after the operation can reach 20%-30%, a large number of animal deaths will lead to the difficulty in comparing experimental data of different batches and the inability to guarantee the consistency of the results; after the model is constructed, it needs to wait for several weeks or even months to observe the recovery of the nerve function of the mouse, which greatly prolongs the experimental period; the physiological conditions of different mice have individual differences, which will lead to inconsistent ischemia range and injury degree of the brain of the mice, and the same pathological conditions cannot be ensured for all experimental animals; and the cost of mouse feeding and operation is high, which is not conducive to large-scale research. In comparison, the zebrafish as a model organism has the advantages of small size, fast reproduction, easy observation and low feeding cost, but there is no mature and popular ischemic nerve injury model at present. SUMMARY
[0004] The application provides a method for constructing an ischemic nerve injury animal model, which takes zebrafish larvae as the research object, induces ischemic nerve injury by treating 1mmol / L cobalt chloride aqueous solution for 24h, and constructs a multi-dimensional evaluation system in combination with behavior detection, brain H&E staining and red cell red fluorescence labeling. The construction method in the application can effectively solve the technical defects of high cost, long experimental period and single evaluation system of the existing ischemic nerve injury model (such as a mouse surgical model), and can be used for high-throughput screening and efficacy evaluation of nerve protection drugs.
[0005] To achieve the above object, the application adopts the following technical scheme: A method for constructing an ischemic nerve injury animal model, the specific steps are as follows: Healthy young fish 3 days after fertilization are placed in a six-hole plate, a cobalt chloride solution is added for treatment for 24 hours, and the construction of the ischemic nerve injury animal model is completed.
[0006] The concentration of the cobalt chloride solution is 1mmol / L.
[0007] The healthy young fish are healthy zebrafish young fish.
[0008] The induced completed zebrafish is placed in a 12mm*12mm square for behavior detection, the moving distance, speed and immobile time of the zebrafish are analyzed, the brain of the induced completed zebrafish is subjected to H&E staining and red fluorescence detection, and the number of neuron cells and the brain blood flow are analyzed.
[0009] The present application uses cobalt chloride to construct an ischemic nerve injury model of zebrafish, which is expected to solve many problems of traditional mouse surgery modeling, not only can improve the survival rate of experimental animals, but also can greatly shorten the experimental period and research cost, and the operation process is more simple, and the stability of the constructed model is higher, which provides a new possibility for efficient research of ischemic nerve injury.
[0010] Compared with the prior art, the beneficial effects of the present application are as follows: (1) The present application breaks through the limitations of traditional animal selection and clearly uses zebrafish young fish as the research object for the first time, which greatly reduces the experimental cost and time cost.
[0011] (2) The present application combines “behavior function (dynamic living body) + histopathology (cell damage) + hemodynamics (ischemic core index) ” to form a standardized multi-dimensional evaluation system, and the repeatability and reliability of the model are greatly improved.
[0012] (3) The present application solves the technical bottleneck that the traditional animal model cannot be applied in high throughput, and provides an efficient tool for the research and development of neuroprotective drugs. BRIEF DESCRIPTION OF DRAWINGS
[0013] Figure 1 is the moving distance of zebrafish after 0, 12, 24, 36 and 48 hours of treatment with 1mmol / L cobalt chloride solution; Figure 2 is the behavior trajectory graph and average heat map of zebrafish after 24 hours of treatment with 1mmol / L cobalt chloride solution; Figure 3 is the quantification graph of the moving distance, moving speed and immobile time of zebrafish after 24 hours of treatment with 1mmol / L cobalt chloride solution; Figure 4is the brain H&E staining chart and neuron cell quantification chart of zebrafish larvae after 24h treatment with 1mmol / L cobalt chloride solution; Figure 5 is the brain red blood cell fluorescence chart and red blood cell fluorescence quantification chart of zebrafish larvae after 24h treatment with 1mmol / L cobalt chloride solution. DETAILED DESCRIPTION
[0014] In order to make the purpose, technical scheme and advantages of the present application more clear, the specific embodiments of the present application are described in detail below, and the examples of the embodiments are shown in the accompanying drawings. The technical scheme of the present application is further described below in combination with the accompanying drawings and specific embodiments.
[0015] The materials, reagents and the like used in the embodiments can be obtained from commercial channels unless otherwise specified.
[0016] The cobalt chloride used in the present application is purchased from the market, AB strain and CZ64 sd2Tg, Tg (gata la :DsRed) The AB strain and CZ64 strain zebrafish are purchased from the National Zebrafish Resource Center.
[0017] The materials and instruments involved in the animal experiments of the present application are as follows: 1. Animals Zebrafish larvae: AB strain and CZ64 strain experimental zebrafish are naturally mated with a female: male ratio of 1:2 to produce eggs, and the fertilized eggs can be used for pharmacological and toxicological experiments after 3 days of collection.
[0018] 2. Drugs and main reagents Cobalt chloride is purchased from Shanghai Yenn Chemical Technology Co., Ltd.; 60x E3 water preparation method: weigh 696mg NaCl, 32mg KCl, 87.56mg CaCl2, and 195.6mg MgCl2·6H2O respectively, dissolve in ultrapure water, and then dilute to 40mL, store at -20℃; 1x E3 water: dilute 60x E3 water with ultrapure water according to the required volume.
[0019] 3. Main test instruments Zebrafish breeding system: Beijing Aisheng Science and Technology Development Co., Ltd.; HPG280BX light incubator: Harbin Donglian Electronic Technology Development Co., Ltd.; 6-well plate, 96-well plate: Wuxi Naisi Technology Co., Ltd.; ANY-maze animal behavior analysis software: American Stoelting Company; 1.5mL EP tube: Nantong Pardaman Experimental Equipment Co., Ltd.; Camera: Sony Co., Ltd.; Cryostat: Thermo Fisher Scientific; Adhesion glass slide: Seerv Biological Technology Co., Ltd.; Pathological sectioning machine: Shanghai Leica Instrument Co., Ltd.; Tissue spreading machine: Zhejiang Kedi Instrument and Equipment Co., Ltd.; Dehydrator: DIAPATH Company; Embedding machine: Wuhan Junjie Company; Integrated fluorescence microscopic imaging system: Keyence Company.
[0020] Example 1 A method for constructing an animal model of ischemic nerve injury, the specific steps are as follows: 1. Zebrafish breeding Adult wild-type AB strain and CZ64 (AB;cz64) strain purchased from the National Zebrafish Resource Center sd2Tg, Tg (gata la :DsRed) Figure 1 The zebrafish of the AB strain is bred in a breeding system with a temperature of 28°C, a pH of 6.8-7.0, and a light-dark cycle of 14h light and 10h dark. Male and female zebrafish are bred separately, and the tanks are divided according to the living conditions of the zebrafish to ensure appropriate density. Adult zebrafish are fed twice a day (once at 9am and once at 5pm), and zebrafish of 1-2 months of age are fed three times a day (once at 9am, once at 2pm, and once at 6pm).
[0021] 2. Zebrafish egg cultivation After feeding the zebrafish for half an hour, the zebrafish for breeding are selected and placed in a breeding box (female: male = 1:2) overnight. The next morning, check the spawning of the zebrafish. When there are a large number of fish eggs at the bottom of the breeding box and the fish no longer chase their tails, the fish are removed for normal feeding, and the fish eggs at the bottom of the breeding box are collected (fertilized fish eggs are transparent, and unfertilized fish eggs are white). The selected fertilized eggs are rinsed with E3 water three times and placed in a glass culture dish for incubation in a 28°C incubator. The water is changed twice a day during incubation, and the white fish eggs are removed during water change. Zebrafish can hatch into juvenile fish about 48-72h after fertilization. The newly hatched juvenile fish do not need to be fed for the first few days after hatching.
[0022] 3. Zebrafish grouping and preparation of cobalt chloride solution Healthy zebrafish juveniles 3 days after fertilization are selected and placed in a 10cm glass culture dish. Each group of 30 fish is randomly divided into four groups and placed in a six-well plate. Deionized water is used to prepare a 100mmol / L cobalt chloride stock solution, which is diluted with E3 water to prepare four concentrations of cobalt chloride solution (0, 0.5, 1, and 2mmol / L), which are prepared fresh and used immediately.
[0023] 4. Determination of optimal cobalt chloride induction concentration Add different concentrations of cobalt chloride solution to the six-well plate at 5mL / well. Then, time and observe the state and movement of the zebrafish juveniles. By comparing the survival time and movement of the zebrafish in each group, the appropriate induction concentration is determined.
[0024] 5. Determination of optimal cobalt chloride induction time With 1 mmol / L cobalt chloride as the inducing concentration, the zebrafish larvae were placed in a 12 mm x 12 mm white hole after being induced for 12, 24, 36 and 48 h, and water was removed as much as possible, and an equal amount of E3 water was added to each hole (flushing with the hole surface), and after 10 min of adaptation, the movement for 30 min was photographed with a camera (the shooting environment required to be quiet), and the movement of the zebrafish larvae was analyzed by using ANY-maze animal behavior analysis software, and the moving distance was mainly analyzed.
[0025] 6. Induction of ischemic nerve injury zebrafish According to the optimal inducing concentration determined in step 4 and the optimal inducing time determined in step 5, 3 dpf healthy zebrafish larvae were randomly divided into two groups and placed in a six-well plate, which were blank control group and cobalt chloride induction group respectively, the blank control group was added with E3 water, and the cobalt chloride induction group was added with 1 mmol / L cobalt chloride solution, 5 mL per hole, and the liquid was changed every 12 h, and the ischemic nerve injury zebrafish model was obtained after 24 h, the zebrafish larvae after induction were placed in a 12 mm x 12 mm white hole, and water was removed as much as possible, and an equal amount of E3 water was added to each hole (flushing with the hole surface), and after 10 min of adaptation, the movement for 30 min was photographed with a camera (the shooting environment required to be quiet), and the movement of the zebrafish larvae was analyzed by using ANY-maze animal behavior analysis software, and the moving distance was mainly analyzed.
[0026] 7. Detection of brain neurons of ischemic nerve injury zebrafish According to the method of step 6, the zebrafish larvae after induction were subjected to H&E staining, and the changes of brain neurons of the zebrafish after induction were analyzed by brain staining results, and the operation steps of H&E staining were as follows: (1) Sampling: the zebrafish larvae in each experimental group were collected in a 1.5 mL EP tube, and washed with E3 water for 3 times; (2) Fixation: the excess water in the EP tube was absorbed, and 4% paraformaldehyde fixing solution was added for fixation at 4°C for 48 h; (3) Gradient dehydration: after the fixation was completed, the zebrafish larvae were washed with water for 10 min to remove the residual fixing solution on the surface, then the larvae were placed in a tissue frame, and dehydrated with 70% ethanol aqueous solution, a mixture of ethanol, n-butanol and distilled water with a volume ratio of 1:1:1, a mixture of ethanol and n-butanol with a ratio of 1:1, and 100% n-butanol; (4) Wax immersion and embedding: after dehydration, the zebrafish larvae were immersed in a mixture of n-butanol and paraffin (n-butanol and paraffin with a volume ratio of 1:1) for 1 h (the temperature of the mixture was 60°C), and the operation was repeated twice, and embedding was immediately performed after wax immersion; (5) Slicing and drying: using a microtome to cut the wax-embedded tissue into small pieces with a thickness of 4-7 microns, and then drying the pieces; (6) De-waxing: sequentially immersing the slices in xylene for 20 min, anhydrous ethanol for 5 min, anhydrous ethanol for 5 min, and 75% ethanol aqueous solution for 5 min; (7) Staining: immersing the slices in a hematoxylin staining solution for 3-5 min, then rinsing with flowing tap water to remove the floating color, then immersing the slices in a 1% acidified ethanol solution (2 mL of 1% hydrochloric acid in 300 mL of ethanol, and then adding 100 mL of distilled water) for 5-10 s, then rinsing with flowing water to stop the reaction, then immersing the slices in a bluing solution to return the color, then rinsing with flowing water, then dehydrating the slices in 95% ethanol for 60 s, and then immersing the slices in an eosin staining solution for 15 s; (8) Recording and observation: after the slices are dehydrated and mounted by immersing them in anhydrous ethanol, observing the slices under a body microscope and collecting images.
[0027] 8. Detection of blood flow in the brain of zebrafish with ischemic nerve injury After the induction is completed according to step 6, the zebrafish larvae, which are labeled with red fluorescent red blood cells, i.e., the CZ64 strain, are moved into a 96-well plate, a small amount of E3 water is added to ensure that the larvae will not die during the observation period, and the fluorescence intensity of the brain of the zebrafish larvae is observed under a fluorescence microscope.
[0028] 9. Data processing and statistical analysis The number of neurons in the brain and the fluorescence intensity of the brain are quantified using ImageJ software; the behavioral data results, the quantification of the number of neurons in the brain, and the quantification of the fluorescence intensity of the brain are statistically analyzed and plotted in Graphpad Prism 9.0 software, and the statistical difference results of the blank control group and the cobalt chloride induction group are marked with an asterisk, where * represents P<0.05, ** represents P<0.01, *** represents P<0.001, and **** represents P<0.0001.
[0029] (1) The survival and movement of zebrafish larvae induced by different concentrations and times of cobalt chloride are shown in Tables 1 and Figure 2 As shown in Tables 1 and 2, the movement of the 1 mmol / L cobalt chloride group changed greatly during the induction process. With the increase of the induction time, the moving distance of the zebrafish larvae decreased, and there was a statistical difference after 12 h (**, P<0.01), and a significant difference after 24 h (****, P<0.0001). Combined with Table 1, the zebrafish larvae died at 36 h, and more died at 48 h. Therefore, the optimal induction concentration is determined to be 1 mmol / L, and the optimal induction time is 24 h.
[0030] Table 1
[0031] (2) Behavior analysis: 3dpf healthy zebrafish larvae were randomly divided into two groups, 10 per group, the blank control group was added with E3 water for 24h, and the cobalt chloride induction group was added with 1mmol / L cobalt chloride solution for 24h, and behavior detection was carried out, the experiment was repeated 3 times, and the results were statistically analyzed, and the results are shown in Figure 3 , Figure 2 , Figure 3 is the motion trajectory diagram and the average heat map, compared with the blank control group, the motion of the cobalt chloride induction group is obviously reduced; Figure 3 A is the average moving speed, Figure 3 B is the total moving distance, Figure 4 C is the total immobile time, compared with the blank control group, the moving distance of the cobalt chloride induction group is reduced (****, P<0.0001), the moving speed is slowed down (****, P<0.0001), and the immobile time is prolonged (****, P<0.0001), which indicates that the motion ability of zebrafish larvae after treatment with cobalt chloride solution is significantly reduced.
[0032] (3) Brain neuron detection: after the above induction method is used, H&E staining is carried out, and the results are shown in Figure 5 , compared with the blank control group, the brain of the cobalt chloride induction group is not complete, and the number of neuron cells is reduced (**, P<0.01), which indicates that cobalt chloride solution treatment can cause neuron cell death.
[0033] (4) Brain blood flow detection: after the above induction method is used, the CZ64 strain of zebrafish larvae is used, and the red fluorescence intensity of the brain is observed using a fluorescence microscope, and the results are shown in , compared with the blank control group, the red fluorescence of the brain of the cobalt chloride induction group is enhanced (**, P<0.01), which indicates that after treatment with cobalt chloride solution, the red blood cells in the brain of zebrafish larvae are aggregated, the blood flow in the brain is poor, and cobalt chloride causes cerebral ischemia.
[0034] In summary, the cobalt chloride solution in the embodiment of the present application can induce zebrafish larvae, cause ischemic nerve damage, and indicate that the construction of the ischemic nerve damage animal model is successful, and the model construction method of the present application can solve the technical defects of high cost, long cycle and single evaluation system of the existing model, and can be used for high-throughput screening and pharmacodynamic evaluation of neuroprotective drugs.
[0035] Although some specific embodiments of the present application have been described in detail by way of example with reference to the drawings, it is to be understood that the above examples are intended to be illustrative only and are not intended to limit the scope of the present application, and that various modifications and changes can be made by those skilled in the art to the particular embodiments described without departing from the spirit and scope of the present application. It is intended that the scope of the present application be limited only by the broadest interpretation of the appended claims to be accorded under 35 U.S.C. § 112.
Claims
1. A method for constructing an animal model of ischemic neural injury, characterized by, The specific steps are as follows: After fertilization, the young fish is placed in a six-hole plate, and a cobalt chloride solution is added to treat for 24 hours to complete the construction of the animal model of ischemic nerve injury.
2. The method for constructing an animal model of ischemic nerve injury according to claim 1, characterized in that, The concentration of the cobalt chloride solution is 1mmol / L.
3. The method for constructing an animal model of ischemic nerve injury according to claim 1, characterized in that, The young fish is a zebrafish young fish.